Abstract:

The invention provides novel vaccination strategies based on a prime-boost
vaccination regiment. The inventors have determined improved ways of
boosting an immune response in a patient previously primed or exposed to
a plurality of epitopes. The improved method requires the epitopes in the
boosting phase to be administered individually, i.e. held on separate
peptide constructs.

5. A method of inducing a CD8+ immune response against melanoma-associated
antigens in a subject comprising administering to said subject a DNA
plasmid according to claim 3.

6. A method of inducing a CD8+ immune response against melanoma-associated
antigens in a subject comprising administering to said subject a
recombinant MVA virus according to claim 4.

7. A method according to claim 6 wherein said subject has been previously
administered with a DNA plasmid according to claim 3.

Description:

[0001]The present invention relates to materials and methods for improving
vaccination strategies. Particularly, but not exclusively, the invention
relates to "prime-boost" vaccination protocols in which the immune system
is induced by a priming composition and boosted by administration of a
boosting composition. The invention further relates to novel tetrameric
soluble class I MHC/peptide complexes as a tool for directly monitoring
vaccination regimens and determining novel epitopes.

[0002]Recent advances in our ability to monitor frequency of antigen CTL
responses in ex-vivo assays are rapidly improving our capacity to compare
different vaccination protocols. In particular, the use of tetrameric
soluble class I MHC/peptide complexes (tetramers) provides an opportunity
to greatly accelerate development of new vaccines by allowing rapid and
accurate analysis of human CTL responses1-3.

[0003]It has become clear that heterologous prime-boost vaccination
protocols, based on repeated injections of non-cross reactive vectors
encoding the same antigenic protein, result in strong CTL responses,
probably due to focusing of the immune response towards epitopes
contained within the recombinant target proteins4. Recent results
have demonstrated that combination of priming with plasmid DNA and
boosting with recombinant defective vaccinia virus MVA generate high
levels of specific immunity5-10.

[0004]Alphaviruses have been extensively studied as viral vectors in
vaccination protocols11-15. The replication incompetent alphavirus,
Semliki Forest Virus (SFV), has proven to be capable of inducing
antibodies and CTL directed against the encoded foreign
antigens14,15. The small size of the SFV genome 16 makes this virus
a very attractive vector for vaccination strategies, as expression of a
small number of viral structural proteins maximise the chances of
generating an immune response specific to recombinant proteins, rather
than to viral structural proteins.

[0005]Several studies have demonstrated that viruses and tumour cells
evade specific immune responses by mutating or deleting antigenic
proteins17,18. In order to minimise the generation of virus or
tumour antigen loss variants, vaccine induced immune responses should be
specific to a broad range of different epitopes, possibly encoded by
distinct proteins. This rationale has led to the generation of vaccines
encoding strings of CTL epitopes, aimed at simultaneously expanding CTL
with different specificity. Vaccination of A2 transgenic mice has shown
that multiple epitopes encoded within poly-epitope constructs can each
prime specific CTL, suggesting the feasibility of this approach for
immunotherapy clinical trials19-22. However, due to the technical
limitations of assays for directly monitoring CTL responses in these
mice, evidence is lacking that polyvalent constructs are capable of
expanding CTL of many specificities to effective levels.

[0006]There is hope that poly-epitope vaccines will be capable of inducing
broad based cytotoxic T lymphocyte (CTL) responses in humans. The
administration of a plurality of epitopes is aimed at simultaneously
expanding CTL with different specificity. Although such polyvalent
constructs have proven capable of simultaneously priming CTL of multiple
specificities in animals which is clearly advantageous, it remains
unclear whether they are capable of subsequently boosting each of these
CTL responses to effective levels.

[0007]It is known that some epitopes are more efficient at raising an
immune response than others. Some epitopes may be described as dominant,
i.e. they provoke a strong CTL response, while others may be described as
subdominant in that they provoke a weaker response. However, when trying
to raise an immune response to a broad range of epitopes it is important
that the subdominant epitopes are not overlooked in favour of more
dominant epitopes.

[0008]During the priming stage of a vaccine regimen the more dominant
epitopes provoke a greater CTL response than the weaker epitopes. This
means that after an initial priming event to a plurality of epitopes, the
CTL response is inevitably greater for the more dominant species and
weaker for the subdominant species. However, the present inventors have
found that the situation is made worse when the same plurality of
epitopes is administered as a poly-epitope construct during the boosting
phase. This appears to be true even when the poly-epitope is provided in
a different vector/vehicle than the priming phase. The inventors have
found that during the boosting stage, the CTL response to the more
dominant epitopes is increased at a greater rate than the CTL response to
the subdominant epitope, to the extent that the expansion of the CTL
response to the subdominant epitopes is significantly reduced. This means
that the CTLs raised to the dominant epitopes are expanded further at the
expense of the CTLs raised to the subdominant epitopes. As a consequence,
the proportion of CTLs raised to the dominant epitopes is increased
whereas the proportion of CTLs raised to the subdominant epitopes is only
marginally increased. This means that the boosting phase narrows the
immune response by favouring proliferation of CTL expanded in the initial
priming stage.

[0009]The present inventors have surprisingly determined a novel
prime-boost regimen that helps to overcome the potentially negative
effect of the boosting phase on a plurality of dominant and subdominant
epitopes.

[0010]Remarkably, the inventors have found that a broad CTL response can
be more uniformly boosted to effective levels if, following the priming
stage, the epitopes are used individually to boost the response as
opposed to being administered as a single poly-epitope construct. By
boosting with the individual epitopes, the inventors have found that the
CTLs raised against the dominant epitopes are not boosted at the expense
of the CTLs raised against the subdominant epitopes. Rather, they are
boosted equally.

[0011]Specifically, and as exemplified below, the inventors have used DNA
and viral vectors encoding a string of melanoma epitopes, to demonstrate
that prime-boost vaccinations result in the expansion of a narrow CTL
repertoire. At the boosting step the inventors found that CTL competition
for recognition of cells presenting the poly-epitope construct skews the
response towards those CTL expanded more efficiently during priming. In
contrast, the inventors have found that simultaneous expansion of CTL
specific to dominant and subdominant determinants is obtained when APCs
were presenting the epitopes separately during the boosting phase. This
could be accomplished, for example, by injecting a mixture of viruses
each encoding a separate antigen or by injecting a mixture of APC
presenting the epitopes separately.

[0012]Thus, the invention provides a method of inducing a specific but
broad based CTL response to a plurality of epitopes, where poly-epitope
constructs are used in the priming phase of a vaccination regimen but
immunogens encoding or comprising the epitopes individually are used for
the boosting phase.

[0013]The invention also provides a heterologous prime-boost vaccination
regimen where epitopes in the boosting phase are presented to the immune
system in a different way than in the priming stage. This is explained in
more detail below.

[0014]The present invention arises from the determination that the
boosting phase is considerably more effective in inducing a specific but
broad based CTL response to a plurality of epitopes if those epitopes are
administered individually.

[0015]Thus, if an individual has already been primed by a plurality of
epitopes, the invention provides a method of boosting the previously
induced (primed) immune response comprising administering the plurality
of epitopes individually, i.e. separately, on separate constructs or
carried by separate vehicles.

[0016]In all aspects of the invention described herein, a plurality of
epitopes may be taken to any number of epitopes greater than 2, more
preferably, greater than 4 and still more preferably greater than 7. Of
the plurality of epitopes, at least two, more preferably four and even
more preferably seven epitopes will be different i.e. comprise a
different amino acid sequence or be recognised by different antibodies.
However, it is also preferably that the plurality of epitopes comprise
many epitopes in the order of tens or hundreds. It is likely that some of
these epitopes will be very similar and may cross-react.

[0017]Thus, if an individual has been infected by, or in some way come in
contact with i.e. been exposed to, a pathogen (e.g. virus, bacteria etc)
or a tumour, then the individual's immune system will have been naturally
primed against a plurality of epitopes presented by the pathogen or
tumour. However, this initial priming of the immune system may well be
insufficient for the individual to mount an effective defense against the
pathogen or tumour. However, in accordance with the present invention,
the already primed immune response may be, boosted by administering the
plurality of epitopes individually, i.e. separately, carried by separate
constructs (peptide or nucleic acid) or separate vehicles. Thus, in this
context, the present invention has vast therapeutic potential.

[0018]Although it would be possible to detect whether an individual had
been primed to a particular epitope, i.e. through exposure to a pathogen
or as a result of a tumour, this step would not be necessary.

[0019]In order to save time, cost and trouble, it would be preferable to
treat every patient suspected of having been primed to a plurality of
epitopes by exposure to a pathogen or a tumour as a primed patient.

[0020]Accordingly, in a first aspect of the present invention, there is
provided a method for boosting an immune response in an individual, said
individual having been previously primed against or exposed to at least
one of said plurality of epitopes, preferably all of said plurality of
epitopes, said method comprising administering to the individual a
plurality of constructs, each encoding or comprising one of said
plurality of epitopes. The construct may be a nucleic acid sequence
capable of encoding a peptide comprising the epitope in question or it
may be a peptide or protein/polypeptide comprising the epitope which can
be administered directly. The nucleic acid sequence may be DNA, RNA or
cDNA capable of encoding a peptide comprising one or more of the epitopes
in question. Whether the construct is nucleic acid sequence encoding the
peptide or a peptide itself, it is preferable to use a vehicle to carry
the construct so that is can be efficiently presented to the individual's
immune system. Preferably, the constructs are each presented or carried
by separate vehicles such as a nucleic acid expression vector, e.g. a
viral vector, or APC, e.g. dendritic cells or lymphocytes e.g. B cells.
The vehicles may also act as adjuvants to help with the inducing immune
response. The APCs may be used to express peptide constructs or they may
be pulsed prior to administration.

[0021]The method in accordance with the first aspect of the present
invention, may further comprise administering a "second" or further
boosting composition. This composition will also comprise individual
constructs, each comprising one of said plurality of epitopes. Again, the
constructs may be peptides or nucleic acid sequences capable of encoding
said peptides.

[0022]The administration of a second or further boosting composition has
the benefit of not only further boosting the individuals immune response
to the administered epitopes but also providing a "first" boost to any
epitopes present in the medicament that had not been already primed
naturally by the individual through exposure to the pathogen or tumour.
In other words, the second/further boosting composition ensures that any
epitopes presented in the medicament ("first" boosting composition) that
the individual had not previously been exposed to, would effectively be
boosted for the first time.

[0023]Alternatively, the present invention may be used to immunise an
individual against a pathogen or tumour associated antigen, i.e. as a
preventative vaccine. In this situation, the individual's immune system
must first be primed by a plurality of epitopes characteristic of the
pathogen and then boosted to help the immune system raise an effective
defense against the pathogen. This is known as a prime-boost regimen.
However, as described above, the inventors have found that to maximise
the immune response against each and every one of the epitopes, the
epitopes must be administered individually in the boosting stage.
Further, the inventors have also determined that a heterologous
prime-boost regimen is preferable to the homologous prime-boost methods
already described in the art. Thus, it also preferable to administer the
plurality of epitopes during the boosting stage using separate vehicles,
e.g. viral vectors, that are different to and non cross-reactive with
vehicles which may have been used in the priming stage.

[0024]Thus, in a second aspect of the present invention, there is provided
a method of inducing an immune response, preferably a CD8+ T cell immune
response, to a plurality of epitopes in an individual, said method
comprising the steps of administering to the individual a priming
composition comprising a construct encoding or comprising said plurality
of epitopes and then administering a boosting composition which comprises
a plurality of individual constructs each comprising one of said
plurality of epitopes.

[0025]In accordance with the second aspect, the construct may be a peptide
(protein/polypeptide) or a nucleic acid sequence encoding said peptide.
it is also preferably, that the constructs are administered using a
vehicle capable of efficiently displaying the epitopes to the
individual's immune system. Thus, where the construct is a nucleic acid
sequence, this may be contained within a nucleic acid expression vector,
i.e. a plasmid or viral vector. These vectors may likewise be contained
within a cell such as an Antigen Presenting Cell (APC).

[0026]Where the construct is a peptide, it may be preferable to use a
cell, more preferably an APC as a vehicle as these cells are capable of
displaying peptides efficiently to the immune system. Examples of APCs
include dendritic cells and lymphocytes.

[0027]The administration of the constructs to an individual using vehicles
is described in more detail below.

[0028]The priming composition may comprise one or more nucleic acid
vectors, each containing nucleic acid encoding a plurality of epitopes.
Alternatively, the priming composition may comprise peptides or antigens
containing a plurality of epitopes.

[0029]In this second aspect, the priming composition comprises a string of
epitopes, i.e. a polyepitope construct. However, the method may
alternatively include the administration of one or more constructs
encoding or comprising one or more of the plurality of epitopes. However,
in this situation, it is preferable that the prime-boost regimen is a
heterologous prime-boost regimen. In other words, if the priming
composition comprises individual epitopes, the boosting composition
preferably carried or presents its individual epitopes using different
and non-cross reactive vehicles.

[0030]For example, where nucleic acid constructs are used different viral
vectors may be used in the priming and boosting phase. Likewise, for
peptide constructs, different APC cells may be used between the priming
and boosting phase, e.g. B cells for priming and dendritic cells for
boosting.

[0031]This is a preferred embodiment of the invention and it must be
appreciated that homologous prime-boost prime-boost regimens are also
within the scope of the present invention, particularly when using
peptide constructs.

[0032]It is also preferred that all priming nucleic acid constructs are
recombinant constructs for example, any genetic constructs like
recombinant viral constructs, DNA constructs, RNA constructs, or cells
transfected or transduced with such constructs. Further the priming
composition may comprise separate peptides or proteins and cells that are
extra- or intracellularly loaded with such peptides or proteins. The
peptides may form part of a fusion construct with a carrier protein or
adjuvant. These may be produced as fusion proteins.

[0033]Where the boosting or priming compositions comprise peptides or
proteins, these may be delivered using Antigen Presenting Cells (APCs)
for example dendritic cells or lymphocytes (B cells), pulsed with peptide
and/or protein (including intracellular delivery of peptides or proteins
into the APC). The APCs once pulsed with peptide or protein may also be
infected by virus in order to help activate the APC, i.e. the virus in
this case acts as an adjuvant for the peptide.

[0034]Particles such as sepharose beads or chitine beads may also be used
to mimic APCs and display peptide/MHC complexes and co-stimulatory
molecules for stimulation of the immune system.

[0035]Exosomes or other subcellular bodies derived from APCs may also be
pulsed with peptide and/or protein (including intracellular delivery of
peptides or proteins into the exosomes) for delivery of the peptide
epitopes.

[0036]It is also possible to administer the peptide or protein directly
into the individual preferably at separate locations. The peptide or
protein administered in this way is preferably accompanied by an adjuvant
in either the priming or boosting phases.

[0037]Where a nucleic acid vector is provided as a vehicle, it is
preferable that the nucleic acid encoding the epitopes is operably linked
to regulatory sequences for production of said antigen in the individual
by expression of the nucleic acid.

[0038]As the priming composition presents a plurality of epitopes, a broad
but specific CTL response is induced by the individual's immune system.

[0039]In contrast to the priming composition, the boosting composition
presents the epitopes always individually. This overcomes the problem
determined by the inventors that pre-existing memory CTL responses
significantly reduce CTL response to other epitopes contained within the
same construct during the boosting phase.

[0040]The priming composition if used, or the boosting composition when
using nucleic acid constructs, may further comprise any vehicle for
carrying the nucleic acid construct encoding the epitopes e.g. a viral
vector, such as adenovirus vectors, Herpes simplex virus vectors,
vaccinia virus vector. The viral vector may be a modified,
replication-deficient vector, e.g. modified virus Ankara (MVA), or it may
be an avipox vector e.g. fowlpox, Canarypox and so on. Preferred vectors
include the replication incompetent alphavirus Semliki Forest Virus
(SFV). Other appropriate vectors will be apparent to those skilled in the
art.

[0041]The priming composition may comprise DNA encoding the antigen. The
DNA may be in the form of a circular plasmid that is not capable of
replicating in mammalian cells. Expression of the antigen will preferably
be driven by a promoter active in mammalian cells, e.g. cytomegalovirus
immediate early (CMV IE) promoter.

[0042]The CD8.sup.+ T cell immune response may be primed using a DNA
vaccine, Ty-VLPs or recombinant modified Vaccinia virus Ankara (MVA). In
the examples provided below, the inventors describe embodiments of the
invention using a recombinant MVA naked plasmid DNA vaccinia virus, and
semliki forest virus (SFV) during the priming phase. However, it will be
apparent to the skilled person that other vectors, viral or otherwise,
may equally be used.

[0043]As mentioned above, it is preferable to use a different, non-cross
reactive vehicle, such as a nucleic acid expression vector, e.g. a viral
vector, for displaying epitopes during the boosting phase than that used
during the priming phase. In the examples provided below, plasmid DNA was
used as a vehicle vector during the priming phase and vectors vaccinia,
MVA and/or SFV were used as vehicles during the boosting phase. As
multiple vectors will be used as vehicles during the boosting phase,
these may be the same or different.

[0044]Thus, in accordance with the first and second aspect of the
invention, there is provided a novel vaccination regimen that can be used
as a method of vaccinating an individual against pathogens including self
antigens or tumour antigens. Exemplified below are the use of NY-ESO-1,
Tyrosinase and Melan-A antigens. However, other antigens will be known or
may be determined by the skilled person.

[0045]Further, the invention has an important utility as a vaccination
model for testing and establishing vaccination strategies or regimens.
Thus, the vaccination model allows vaccination regimens to be established
which maximise a specific but broad based CTL response to a plurality of
epitopes.

[0046]It is usual to test vaccine regimens on laboratory animals such as
mice prior to testing on humans in clinical trials. Transgenic mice have
been used in the past to test responses to particular antigens or
epitopes. It is important to provide a biological environment that is as
equivalent to the human environment as is possible. Therefore, transgenic
mice are produced which have the ability to express human MHC molecules.
In the examples provided below, the inventors have used two forms of
transgenic mice.

[0047]Firstly, they have used HHD A2 transgenic mice which express a
transgenic monochain histocompatibility class I molecule in which the C
terminus of the human β2m is covalently linked to the N terminus of
a chimeric heavy chain (HLA-A2.1 α1-α2, H-2Db α3
transmembrane and intracytoplasmic domains). The H-2Db and mouse
β2m genes of these mice have been disrupted by homologous
recombination resulting in complete lack of serologically detectable cell
surface expression of mouse histocompatibility class I molecules.

[0049]Thus, both of these transgenic mice express a chimeric MHC where the
α1 and α2 domains are derived from human A2 MHC and the
α3 domain is from murine H-2Kb or H-2Db. These mice are
referred to as A2 transgenic mice. However, the present invention may
equally well be performed on HLA-A1, HLA-A3 or HLA-A4 transgenic mice,
i.e. any other murine model expressing human class I molecule s in a
similar way as A2 transgenic mice do.

[0050]Therefore, the invention further provides a method of testing a
vaccination regime comprising the steps of administering a primary
composition to a test animal, said primary composition comprising a
nucleic acid encoding a plurality of epitopes under test, subsequently
administering a boosting composition, said boosting composition
comprising a plurality of nucleic acid vectors each containing one of the
said plurality of epitopes under test; and determining the CTL response
to each of the epitopes under test.

[0051]Preferably, the test animal will be a transgenic animal that
provides an immune environment as close to the human immune environment
as possible. The preferred test animal is an A2 transgenic mouse as
described herein.

[0052]As discussed above, the priming vector preferably is a DNA plasmid
or viral vector. However, the boosting vectors are preferably different
to the priming vector. In a preferred embodiment the priming vector is
DNA, and the boosting vector is selected from the group of vaccinia, MVA
and/or SFV. SFV is the preferred vector for the boosting phase as the
inventors have determined that excellent results are achieved when used
following DNA priming vector or with MVA as the boosting vector. SFV does
not cross-react with MVA and therefore these immunogen vectors can be
effectively used together. The inventors have also found that the nucleic
acid constructs whether as part of a viral vector or not, may be
administered in association with a mammalian cell, such that the antigens
are presented on its surface. Such cells are known as Antigen Presenting
Cells (APCs) when antigen is presented on their surface. Thus, the term
APC includes cells such as tumour cells. The use of APCs to carry the
construct is particularly preferable during the boosting stage. Thus,
possible boosting reagents include APCs, such as dendritic cells or
lymphocytes transfected or transduced with the viral or non-viral nucleic
acid construct or tumour cells displaying antigen.

[0053]In particular embodiments of the first aspect of the present
invention, administration of a priming composition is followed by
boosting compositions, the priming composition and the boosting
composition being different from one another, e.g. as exemplified below.
However, a second and third boosting composition may be administered
within the method of the present invention as mentioned above. In one
embodiment, a triple immunisation regimen employs DNA expressing a
plurality of epitopes; followed by SFV as a first boosting composition
where a plurality of SFV vectors are used each comprising one of the
plurality of priming epitopes; followed by MVA as a third boosting
composition where a plurality of MVA vectors are used each comprising one
of the plurality of priming epitopes. Alternatively, the SFV boosting
composition may be administered after the MVA boosting composition.

[0054]Likewise, where peptides are administered directly, peptide pulsed
APCs, exosomes or APC mimics may also be used for several sequential
boosting steps. This may be achieved by sequential injections.

[0055]In all cases, it is preferable to administer (e.g. by injection) the
priming and boosting composition several times to ensure successful
delivery.

[0056]Priming with vectors expressing a plurality of epitopes can be
followed by a mixture of recombinant virus (i.e. either SFV or MVA)
comprising one or more of the plurality of priming epitopes and followed
by a further boosting based on the injection of peptides, each, encoding
one of the plurality of priming epitopes. The peptides will preferably be
between 5 and 15 amino acids in length, more preferably between 5 and 12
amino acids in length, more preferably between 5 and 10 amino acids in
length. 9 amino acid long peptides are preferred. The peptides may be
naturally occurring or they may be synthetic.

[0057]In order to enhance the method of testing vaccination regimens or
strategy, the inventors have appreciated that accurate, efficient and
fast testing of the provoked CTL response is required. With this is mind,
the present inventors have developed a novel tetramer based technique to
directly monitor the frequency of HLA A2 restricted CTL expanded in
vaccinated HLA A2 transgenic mice. In association with the first aspect
of the present invention, this will greatly accelerate development of new
vaccines by allowing rapid and accurate analysis of human CTL responses.

[0058]Specifically, the inventors have developed a technique for directly
monitoring A2 restricted CTL responses in the blood of A2 transgenic mice
by engineering chimeric A2 class I molecules containing the mouse
H2Kb alpha 3 domain. This technique allows accurate monitoring of
the frequency of CTL induced by prime-boost regimens using poly-epitope
constructs encoded within a number of different vectors and the
correlation of the frequency of these CTL with their cytotoxic activity
in vivo. This has the advantage of reducing the number of mice in each
study considerably as they do not have to be killed for their CTL
responses to be addressed.

[0059]Thus, in a further aspect of the invention, there is provided a
multimeric MHC structure that is capable of detecting specific CTLs
expanded following vaccination of an individual or test animal with one
or more epitopes. The multimeric MHC structure comprises two or more MHC
molecules, preferably four molecules, held together in a single structure
by a binding member such as streptavidin. The streptavidin used here may
be fluorescently labelled for the detection of the CTLs binding the
tetramer. As a plurality of MHC molecules are held in close proximity,
they can by effectively used to display the epitope of interest in the
form of a peptide so as to detect CTLs raised to that epitope. If
effectively primed, the CTLs will recognise the displayed peptide/epitope
and bind to the structure. This binding can be detected by known
techniques such as flow cytometry, usually in combination with labelled
antibodies e.g. anti. CD8 antibodies.

[0060]However, the inventors have found that the use of these multimeric
MHC structures, preferably tetramers, for testing the CTL responses in
the vaccination model described above is limited as the MHC is of human
origin whereas the test animal will be non-human, usually mouse, and
therefore will have murine CD8 which does not effectively binding to
human MHC.

[0061]To overcome this problem, the inventors have devised a chimeric
multimeric MHC structure where the human α3 domain of the MHC is
replaced with a murine α3 domain.

[0062]As the α3 domain binds the CD8 molecule, the chimeric
multimeric MHC structure is more efficient at detecting the CTL response
to the epitope under test than the non-chimeric structure.

[0063]Therefore, the invention further provides a chimeric multimeric MHC
structure comprising at least two human MHC molecules held in close
proximity by a binding member, wherein each MHC molecule contains an
altered α3 domain so as to represent a murine α3 domain
instead of a human α3 domain. Preferably, the human α3 domain
is replaced by a murine α3 domain. The chimeric multimeric MHC
structure may also be complexed with peptides displaying the epitope
under test.

[0064]The murine α3 domain may be inserted into the human MHC to
replace the human α3 domain, by creating a fusion protein, or by
mutating the human α3 domain by insertion, deletion, or
substitution of amino acids or nucleotides encoding amino acids
characteristic of the murine α3 domain. Ideally, the chimeric MHC
is produced as a fusion protein where nucleic acid encoding the human
α1 domain and human α2 domains is expressed along with
nucleic acid encoding the murine α3 domain. In this way a chimeric
MHC fusion protein will be produced.

[0065]The multimeric chimeric MHC structure can then be associated with a
peptide displaying the epitope under test. The number of peptides being
displayed by the chimeric MHC will depend on the number of MHC molecules
in the multimeric structure. The inventors have produced tetramers which
will allow four peptides/epitopes to be displayed in close proximity.
Thus, this structure can be used to detect the presence of a CTL response
to the epitope in question. For example, the tetramer will display the
peptide/epitope and when added to a biological sample (e.g. blood)
obtained from the test animal, any CTLs recognising the epitope will bind
to the tetramer with the aid of murine CD8. As the murine CD8 binds more
successfully to murine α3 domain than human α3 domain, the
binding of the CTL to the chimeric MHC tetramer according to the present
invention is more stable than binding to a non-chimeric MHC tetramer.

[0066]The binding of the tetramer to the CTL can be detected by using, for
example a fluorescently labelled tetramer. In addition, labelled CD8
antibody can be used to aid in the detection labelling and staining
techniques are known to the skilled person.

[0067]As well as being extremely useful in monitoring the CTL response in
accordance with the vaccination regimens mentioned above, the chimeric
multimeric MHC structures can also be used to quickly and efficiently
determine epitopes in a particular protein.

[0068]For example, A2 transgenic mice immunised with defined tumour or
viral antigenic protein(s) encoded by DNA and/or recombinant viruses can
be monitored for their ability to mount a specific CTL response by using
the chimeric multimeric MHC class I molecules associated with peptides
derived from the antigenic protein(s). This protocol will make possible
the rapid identification of novel peptide epitopes encoded within
antigenic proteins.

[0069]The inventors' findings are of importance for the design of
optimised vaccines capable of simultaneously expanding high numbers of
CTL specific for multiple epitopes. They are also important with regard
to providing a vaccination model which allows quick, efficient and
reliable testing of epitopes and allows design of the most efficient
vaccination regimen.

[0070]The inventors have also devised a novel chimeric MHC multimer which
can be used to efficiently detect a CTL response to a test epitope in a
test animal.

[0071]Aspects and embodiments of the present invention will now be
illustrated, by way of example, with reference to the accompanying
figures. Further aspects and embodiments will be apparent to those
skilled in the art. All documents mentioned in this text are incorporated
herein by reference.

[0076]FIG. 3 Prime-boost of A2/Kb mice with DNA.mel3 followed by MVA.mel3.

[0077]A. Simultaneous generation of Db and A2 restricted CTL in A2/Kb
transgenic mice. Mice were primed i.m. with DNA-mel3 and boosted 10 days
later i.v. with MVA-mel3. Ex-vivo tetramer analysis of DB/NP366-374 and
A2/melan-A 26-35 was carried out. Frequency of tetramer positive cells is
shown in each vaccinated mouse after 3 days from MVA boost.

[0078]B. Effect of pre-existing memory CTL response specific to a single
determinant contained within the poly-CTL epitope construct. A2/Kb mice
were immunised i.n. with influenza virus and subsequently injected with
DNA.mel3 followed by MVA.mel3. Ex-vivo tetramer analysis of Db/NP366-374
and A2/melan-A 26-35 was carried out. Frequency of tetramer positive
cells is shown in each vaccinated mouse after 3 days from MVA boost.

[0080]FIG. 5 Immunodominance of Melan-A specific CTL response can be
overcome by poly-vaccinia-boosting or by adoptive transfer of in vitro
infected splenocytes. DNA.mel3 primed HHD mice were either boosted with a
mixture of vaccinia viruses encoding the full length tyrosinase and full
length NY-ESO-1 (A). Alternatively, DNA mel3 primed HHD mice were
injected either with three aliquotes of splenocytes separately infected
in vitro with full length tyrosinase, full length NY-ESO-1 and mel3.
Vaccinia (B, panels a, b and c) or with splenocytes infected with mel3
vaccinia (B, panels d, e and f). Frequency of melan-A, tyrosinase and
NY-ESO-1 specific responses were simultaneously measured by ex-vivo
tetramer staining.

[0081]FIG. 6 Poly-virus boosting overcomes the immunodominance of
melan-A26-35 specific CTL. DNA.mel3 primed HHD mice were boosted
with either a mixture of vaccinia viruses encoding the full length
tyrosinase, full length NY-ESO-1 and SFV.mel3 (A) or with SFV.mel3 (B).
Frequencies of melan-A26-35, tyrosinase369-377 and
NY-ESO-1157-165 specific responses were simultaneously measured by
ex-vivo tetramer staining. Staining of a single mouse out of six is
shown. C and D: Each mouse was injected with fluorochrome labeled
splenocytes pulsed with either the melan-A26-35,
tyrosinase369-377 or NY-ESO-1157-165 peptide and the % of in
vivo lysis was calculated. Panel C corresponds to the in vivo killing in
DNA.mel3 primed HHD mice boosted with a mixture of vaccinia viruses
encoding the full length tyrosinase, full length NY-ESO-1 and SFV.mel3,
while panel D corresponds to the in vivo killing in DNA.mel3 primed HHD
mice boosted with SFV.mel3.

[0082]FIG. 7 The experiment shows A2Kb tetramer stains from mice
primed with MVA.mel3 and boosted with peptide pulsed dendritic cells
(DCs). Two groups of mice are shown: Group A (three mice) received DCs
pulsed with a mixture of 3 peptides (Melan-A, Tyrosinase and NY-ESO-1),
and Group B (four mice) received a mixture of DCs pulsed with single
peptides. On the left hand side of the figure, one individual response to
three epitopes is shown from one mouse in each group. On the right hand
side of the figure the average percentage of tetramer positive CTL in
each group is shown. The error bars indicate the standard deviation of
the mean. The experiment shows that peptide pulsed DCs can efficiently
boost poly valent CTL responses primed by recombinant MVA (compare FIG.
4A, B and C) The experiment further demonstrates, that a mixture of
separately peptide pulsed DCs for boosting is more efficient in boosting
a poly-valent response when compared to DCs pulsed with a mixture of
peptides.

DETAILED DESCRIPTION

Materials and Methods

Plasmid DNA Construct

[0083]The DNA vector pSG2, used throughout the study, was derived from
pRc/CMV (Invitrogen, Paisley, UK) by removing the BamH1 fragment that
contains the SV40 origin of replication and neomycin resistance marker
and replacing the CMV promoter with a longer version of the same promoter
containing intron A. The resulting plasmid contains the CMV promoter with
intron A for expression in eukaryotic cells, followed by a multiple
cloning site and the bovine growth hormone poly-A sequence. The plasmid
is incapable of replication in mammalian cells. The gene encoding the
mel3 sequence (Table 1) was introduced into the multiple cloning site
using standard methods. Plasmid DNA for injection was purified using
anion-exchange chromatography (Qiagen, Hilden, Germany) and diluted in
phosphate buffered saline (PBS) at 1 mg DNA/ml.

Generation of Recombinant Vaccinia Virus and MVA

[0084]Recombinant and non-recombinant MVA were routinely propagated and
titrated in chicken embryo fibroblasts (CEF) grown in minimal essential
medium supplemented with 10% foetal calf serum (FCS). Recombinant MVA
were made as described by cloning the mel3 poly-epitope string (Table 1)
into the vaccinia shuttle vector pSC11. CEF infected with MVA at a
multiplicity of 0.05 pfu per cell were transfected with lipofectin
(Gibco) and shuttle plasmid as described23. The Vaccinia P7.5
promoter drives expression of the polyepitope. Recombinant MVA were
plaque purified 8 times.

[0085]Vaccinia viruses (WR strain) expressing mel3, full length NY-ESO-1
(kindly provided by Dennis L. Panicali, Therion Biologics Corporation, MA
02142, USA) or tyrosinase were made by cloning the mel3 poly-epitope
construct, the NY-ESO-1 and tyrosinase full length cDNA into the
thymidine kinase gene using the vector pSC11 as previously
described24.

Generation of Recombinant SFV

[0086]The mel3 poly-epitope string was cloned into the transfer vector
pSFV4.2-mel3. RNA produced from this vector was used to construct
recombinant SFVmel3 particles. Recombinant SFV stocks were made and
purified as described previously 16.

[0088]Fluorescent tetrameric HLA-A2.1/peptide complexes were synthesised
as previously described1. A2-Kb/peptide complexes were synthesised
in an analogous fashion using a chimeric heavy chain of α1 and
α2 domain of the A2.1 molecule and the α3 domain, of the H-2
Db molecule with human β2-micro globulin.

[0090]HHD mice express a transgenic monochain histocompatibility class I
molecule in which the C terminus of the human β2m is covalently
linked to the N terminus of a chimeric heavy chain (HLA-A2.1
α1-α2, H-2Db α3 transmembrane and intracytoplasmic
domains) 20. The H-2Db and mouse β2m genes of these mice have
been disrupted by homologous recombination resulting in complete lack of
serologically detectable cell surface expression of mouse
histocompatibility class I molecules. A2-Kb mice express chimeric
heavy chain (HLA-A2.1 alpha 1 alpha 2, H-2 Kb alpha 3 transmembrane
and cytoplasmic domains) in non-covalent association with mouse β2m.
They additionally express a full set of C57BL/6-derived (H-2b) class
1a and 1b mouse histocompatibility molecules26. All A2 transgenic
mice used were bred in the inventors' animal facility. Female C57/BL6
mice 4-6 weeks old were obtained from Harlan Orlac (Shaws Farm,
Blackthorn, UK). Plasmid DNA (25-50 μg/muscle) was dissolved in PBS
and injected into each musculus tibialis under general anaesthesia. 10
days after DNA injection, mice were boosted with recombinant vaccinia
viruses, which were diluted in PBS and 106-107 pfu and injected
intravenously (i.v.) into the lateral tail vein. Alternatively freshly
isolated spleen cells were separately infected (multiplicity of infection
of 5) with mel3, full length tyrosinase and NY-ESO-1 vaccinia for 90
minutes in RPMI supplemented with 0.1% BSA at 37° C. Cells were
washed 3 times and re suspended in sterile PBS at a concentration of
6×107 cells/ml combined and injected into lateral tail vein.
Mice received 6×106 spleen cells.

[0091]Mice primed with influenza virus A (PR8) were infected by
intra-nasal influenza injection (20 HAU/mouse). 30 days later mice were
injected with DNA.mel3 followed by MVA.mel3, as described above. For
priming or boosting with SFV-mel3 108 virus particles were diluted
in sterile PBS and injected into the lateral tail vein.

In Vivo Killing

[0092]Freshly isolated spleen cells from HHD mice were separately
incubated in RPMI medium with different peptides at a concentration of
10-6M for 2 h. Each cell pool was then labelled with a different
concentration of carboxyfluorescein diacetate succinimidyl ester (CFSE,
Molecular Probes, Eugene, Oreg.) to allow simultaneous tracking of the
different populations in vivo 27, Hermans, I. F., Yang, J. and Ronchese,
F. Unpublished results. Labelled cells were pooled and injected at
107 cells/mouse into the tail vein. A control population without
peptide that had been labelled with 5-(and
-6-)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CellTracker
Orange, Molecular Probes, Eugene, Oreg.) was co-injected to assess
killing of peptide pulsed targets relative to unpulsed cells. Mice were
bled at the time of injection of flurochrome labelled targets to
determine their CTL frequencies to different epitopes. Disappearance of
peptide/flurochrome labelled cells was tracked using FACS analysis of
freshly isolated PBMC 5 h after the injection. Percentage killing was
calculated relatively to the unpulsed population labelled with Cell
Tracker Orange. 100-(100×(% pulsed/% unpulsed)). WinMDI 2.8 and
CellQuest 3.3 software was used to analyse the facs data.

Results

[0093]A string of 5 HLA-A2 and 2 HLA-A1 melanoma, epitopes was cloned into
four distinct vectors: a) naked plasmid DNA (mel3. DNA); b) vaccinia
virus (mel3-vaccinia); c) Modified Vaccinia Ankara virus (mel3. MVA); d)
Semliki Forest Virus (mel3-SFV). To ensure monitoring of CTL responses
restricted by human and mouse class I molecules, the inventors introduced
an additional epitope from the influenza Nucleoprotein (NP) restricted by
H-2 Db class I molecules. Since they had previously shown that
presentation of amino-terminal NP366-374 epitope can be affected by
neighbouring amino acid residues 24, the inventors decided to express the
influenza NP 366-374 epitope at the carboxyl terminal end of the
poly-epitope construct. Sequence of the poly-epitope constructs used in
the paper are shown in Table 1.

Efficient Presentation of MVA Encoded Poly-Epitopes Initial experiments
were carried out, to compare the A2 binding affinity of each epitope
contained within the mel.3 construct. The results of these experiments
demonstrated that mel3 peptide epitopes had a broad range of binding
affinities for A2 molecules. The Melan-A 26-35 peptide analogue 28
had the highest binding affinity, while the NY-ESO-1 157-165 and
tyrosinase 1-9 peptides had a significantly lower affinity, as defined by
their ability to inhibit at different concentrations presentation of the
influenza Matrix epitope 58-66 (data not shown). The inventors and others
have previously demonstrated that optimal flanking residues are important
to ensure presentation of class I restricted epitopes 24,29. To establish
that mel.3 peptide epitopes were properly processed, and to assess that
competition for binding to A2 molecules did not impair CTL recognition of
lower affinity epitopes, they infected target cells with mel.3 vaccinia
and demonstrated that each of the 7 epitopes contained within the
poly-epitope mel.3 cassette was simultaneously presented to specific CTL
(FIG. 1). The inventors had previously shown that proteasome dependent
degradation impairs presentation of the MAGE3 A2 epitope 271-27930.
It was therefore surprising to observe that infection of target cells
with mel3 vaccinia was capable of sensitising them for lysis by MAGE3
271-279 specific CTL. Further experiments demonstrated that processing of
the MAGE3 271-279 epitope contained within the mel3 construct, unlike its
processing within the full length MAGE3 protein, was lactacystein
resistant and TAP independent (data not shown), consistent with
processing of mel.3 construct by Endoplasmic reticulum resident
proteases.Influenza NP 366-374 Specific CTL Responses in Mice Vaccinated
with Poly-Epitope Encoded Vaccines

[0094]Efficient presentation of the NP366-374 epitope by mel3 vaccinia
infected cells prompted the inventors to assess in C57/B6 mice the
ability of different vaccination strategies to induce a strong NP366-374
specific CTL response (FIG. 2). Ex-vivo monitoring of the NP specific CTL
response was carried out in PBL using Db/Influenza NP366-374 tetramers.
The inventors compared homologous vs heterologous prime boost vaccination
protocols (FIG. 2), and analysed the kinetics of CTL induction by DNA or
MVA priming (data not shown). The results of these experiments confirmed
that heterologous vaccination strategies are capable of inducing long
lasting vaccine driven CTL responses, with frequencies up to 100 times
greater than frequencies obtained by strategies based on repeated
injections of the same antigen delivery system (FIG. 2).

Expansion of A2 Restricted CTL in A2 Transgenic Mice

[0095]To test the ability of the mel.3 poly-epitope constructs to prime A2
restricted CTL responses in vivo, A2 transgenic mice were primed by
DNA-mel.3 and boosted by MVA-mel.3, vaccinia mel.3, or SFV-mel3. Initial
experiments were carried out using A2.1 transgenic mice, which express
chimeric A2.1 molecules containing the Kb α3 domain, and endogenous
Db and Kb molecules (A2/Kb mice)26. To enable monitoring
of the A2 restricted responses at the same time as the Db restricted
Influenza NP366-374 response, the inventors employed novel A2/Kb
tetramers, which were also capable of detecting the relevant CTL directly
in PBL. Simultaneous staining with A2/Kb and Db tetramers
demonstrated that priming of A2/Kb mice with DNA.mel3, followed by
MVA-mel.3, induced melan-A 26-35 and Influenza NP 366-374 specific CTL
responses (FIG. 3). In contrast, responses specific to other mel.3
epitopes were not detectable by ex vivo tetramer stainings (data not
shown).

[0096]The ability to simultaneously monitor CTL responses against the
influenza NP epitope 366-374 and melan-A epitope 26-35 prompted the
inventors to study whether previous exposure to influenza virus may
compromise the ability of prime-boost protocols to expand melan-A 26-35
specific CTL in A2-Kb mice. In order to generate a strong NP366-374
specific CTL response, A2 transgenic mice were immunised with influenza
virus and subsequently received DNA.mel3 followed by MVA-mel3 (FIG. 3).
The results of these experiments demonstrated that expansion of NP366-374
specific CTL, prior vaccination with mel3 poly-epitope constructs,
significantly reduced the expansion of melan-A specific CTL (FIG. 3). The
inhibitory effect of pre-existing flu specific CTL on the ability of mel3
prime-boost to induce melan-A specific CTL response (FIG. 3) raised the
possibility that T cell interference during heterologous vaccination
strategies may compromise the induction of a broad range immune response.
The presence of endogenous mouse class I molecules significantly narrows
the A2 restricted repertoire in A2 Kb mice, hence hampering the ability
to study the interplay between A2 restricted CTL specific to different
vaccine encoded determinants.

[0097]This reasoning led the inventors to monitor the hierarchy of vaccine
driven CTL responses upon prime boost protocols in the A2 transgenic mice
HHD20. HHD mice, unlike A2Kb transgenic mice, express A2.1 class I
molecules linked to human β-2 microglobulin in a Db-/- and
β-2m-/- background, and have a much larger A2 restricted T cell
repertoire than A2/Kb transgenic mice20.

[0098]Priming of HHD mice with DNA.mel3 led to the expansion of Melan-A
specific CTL to frequencies detectable by ex-vivo tetramer staining in
all vaccinated mice (data not shown). In contrast, expansion of NY-ESO-1
and tyrosinase specific CTL was only detectable in a small proportion of
immunised mice, while responses to the Tyrosinase 1-9 and Mage3 271-279
were not detected in blood tetramer stainings (data not shown).

[0099]Additional experiments confirmed that NY-ESO-1 specific CTL
responses were primed by DNA.mel3 injection, as shown by the significant
NY-ESO-1 CTL response in DNA.mel3 primed mice boosted with vaccinia virus
encoding the full length NY-ESO-1 (data not shown). In contrast,
injection of NY-ESO-1 vaccinia virus, without priming with DNA.mel3, led
to a much lower frequency of NY-ESO-1 CTL. Similar results were obtained
upon injection of tyrosinase vaccinia in mice primed with DNA mel3 (data
not shown).

[0100]The observation that the melan-A 26-35 specific CTL was the dominant
vaccine driven CTL response after a single DNA vaccination, presented an
opportunity to study the interplay between CTL specific to different
vaccine encoded determinants in prime-boost vaccination protocols. The
inventors observed that boosting of DNA.mel3 primed HHD mice with either
vaccinia mel3 (FIG. 4a), MVA.mel3 (FIG. 4b), or SFV-mel3 (FIG. 4c), led
to the expansion of melan-A specific CTL, up to 70-80% of total CD8+ T
cells. Although responses specific to NY-ESO-1 and tyrosinase epitopes
were significantly lower than the Melan-A specific responses, their
frequencies ranged between 2 and 30% of CD8+ T cells, confirming that DNA
priming and boosting with either replication competent (i.e. Vac.mel3) or
incompetent viruses (MVA.mel.3 and SFV-mel3) significantly enhance the
frequency of CTL specific to three distinct melanoma specific epitopes.
The inventors confirmed that vaccine driven CTL were cytolytic, as shown
by their ability to kill fluorochrome labelled splenocytes pulsed with
relevant peptides in vivo (FIG. 4d). These results demonstrated that the
cumulative response specific to melan-A, NY-ESO-1 and tyrosinase in HHD
mice primed with DNA.mel3 and boosted with three distinct viral vectors
accounted for the specificity of the majority of CD8+ population.

Competition of Vaccine Driven CTL for Mel.3 Expressing APC

[0101]Since the inventors demonstrated the inhibitory effect of a
pre-existing flu memory CTL response on the ability to induce melan-A
specific CTL (FIG. 3), they sought to study whether the high numbers of
melan-A specific CTL, dominating the immune response after DNA priming,
were capable of interfering with the expansion of NY-ESO-1 and tyrosinase
specific CTL during the virus boosting.

[0102]It is known that competition for antigen recognition on the surface
of antigen presenting cells leads to the immunodominance of higher
frequency CTL populations31-33. The inventors reasoned that the
higher numbers of melan-A CTL after DNA.mel 3 priming may lead either to
rapid killing or shielding of mel3 vaccinia infected APC in vivo,
resulting in a hampered stimulation of CTL specific to NY-ESO-1 and
tyrosinase epitopes expressed by the same APC population.

[0103]This reasoning led them to assess whether a higher frequency
NY-ESO-1 and tyrosinase specific CTL responses could be obtained by
separating the APC expressing NY-ESO-1 and tyrosinase proteins from the
APC expressing the mel3 construct. The results of these experiments
confirmed this hypothesis, as shown by: 1) expansion of NY-ESO-1 and
Tyrosinase specific CTL in DNA.mel3 primed mice and boosted with a
mixture of vaccinia viruses, encoding the full length tyrosinase and full
length NY-ESO-1 proteins (FIG. 5a); 2) simultaneous expansion of melan-A,
NY-ESO-1 and tyrosinase specific CTL upon adoptive transfer into DNA.mel3
primed mice of three aliquotes of splenocytes infected ex-vivo with
vaccinia viruses encoding full length NY-ESO-1, tyrosinase and mel3
construct (FIG. 5b, panels a, b and c), while adoptive transfer of
splenocytes infected with mel3 vaccinia led to the expansion of Melan-A
specific CTL (FIG. 5b panels d, e and f)

[0104]The Inventors have Demonstrated that Boosting of DNA.Mel3 primed
mice with a mixture of recombinant viruses, encoding the full length
tyrosinase, full length NY-ESO-1 and the mel3 construct, led to the
simultaneous expansion of melan-A26-35, NY-ESO-1157-165
tyrosinase369-377 specific CTL (FIGS. 6a and b). The identification
of successful vaccination strategies to simultaneously expand large
numbers of CTL with a broad specificity has important clinical
applications, as we showed that T cell immunity induced by this type of
optimised boosting strategies provides a more efficient in vivo killing
of target cells than vaccinations based on poly-epitope prime boost
strategies (FIG. 6 c and d).

[0105]Immunodominance of Melan-A specific CTL could be broken by
separating the antigens during the boost. When separately infected
splenocytes were used to boost a polyvalent response relevant peptides
were separately presented and resulted in simultaneous expansion of
Melan-A, Tyrasinase and NY-ESO specific CTL. To simplify this approach
the inventors used peptide pulsed dentritic cells to boost an MVA.mel3
primed response. The cells used for boosting were either pulsed with a
mixture of peptides (FIG. 7a) or separately pulsed (FIG. 7b). The
inventors show that separate pulsing of APC is superior to pulsing APC
with a mixture of peptides. This approach also demonstrates, that
poly-epitope constructs encoded in vaccinia virus can efficiently prime
and APC pulsed with peptide can efficiently boost a polyvalent CTL
response.

Discussion

[0106]There is a tremendous momentum in vaccine development, as recent
advances in the monitoring of antigen specific CTL responses in ex-vivo
assays are rapidly improving our capacity to compare different
vaccination protocols. In order to minimise the generation of tumour and
virus antigen escape variants, it is important to ensure the expansion of
vaccine driven CTL specific to several epitopes, including both dominant
and subdominant determinants. Several papers have dissected the causes
responsible for immunodominance of CTL specific to viral34-36, and
histocompatibility antigens33,37,38. However, it remains to be
established how optimal vaccine strategies can lead to the simultaneous
expansion of CTL specific to dominant and subdominant epitopes.

[0107]To address these questions the inventors engineered 4 distinct
vectors encoding a string of melanoma CTL epitopes (Table 1), and
compared in A2 transgenic and wild type B6 mice the ability of different
vaccination strategies to elicit vaccine specific CTL responses. In order
to identify strategies capable of expanding CTL specific to dominant and
subdominant determinants, peptide epitopes with high and low binding
affinity for A2 molecules were linked in the same construct. More
specifically, the inventors included the modified melan-A analogue 26-35,
previously shown to have an enhanced immunogenicity in vivo28 and
the NY-ESO-1 peptide 157-165, shown to have a much lower binding affinity
to A2 molecules39 (Table 1).

Vaccine Driven CTL Hierarchy in Mel3 Vaccinated Mice

[0108]The inventors have developed a novel tetramer based technique to
directly monitor the frequency of A2 restricted CTL expanded in A2
transgenic mice vaccinated in a prime-boost regimen. To increase the
binding affinity of mouse CD8 to A2 molecules, they engineered A2
molecules containing the mouse H-2Kb alpha 3 domain (A2/Kb
molecules), and demonstrated that tetrameric A2Kb molecules have an
increased staining efficiency for mouse A2 restricted CTL and can
identify A2 restricted responses in a large proportion of A2 transgenic
mice, as compared with tetrameric A2 molecules.

[0109]While studying the immune response in A2Kb mice, the inventors have
demonstrated that expression of Db molecules results in a strong
influenza NP366-374 specific response, which significantly impairs the
expansion of CTL specific to other mel3 encoded epitopes. In order to
study the interplay between A2 restricted CTL specific to different
vaccine encoded determinants, the inventors immunised the A2 transgenic
mice HHD 20, which, unlike A2Kb transgenic mice, express A2 molecules in
a Db-/- background.

[0110]Although several papers have recently studied the immune response in
A2 transgenic mice vaccinated with poly-epitope constructs 19,20,40, this
is the first publication in which the hierarchy of poly-epitope vaccine
driven CTL in A2 transgenic mice has been monitored by ex-vivo tetramer
staining.

[0111]The inventors compared several vaccination strategies and confirmed
that immunisations based on the injection of non-cross reactive vectors
(heterologous prime-boost protocols) ensure higher levels of vaccine
specific immune responses than immunisations based on the injections of
homologous vectors (FIG. 2). The presence of neutralising antibodies
against viral structural proteins and the presence of CTL specific to
viral proteins may account for the lower CTL responses in mice vaccinated
with repeated injections of the same virus, as compared with CTL
frequencies upon vaccination with non-cross reactive vectors (FIG. 2). It
has been suggested that the limited number of proteins encoded by plasmid
DNA ensures that DNA priming focuses the immune response towards the
recombinant protein, while virus boosting successfully expand this
response, resulting in high levels of CTL specific to the recombinant
protein. However, the inventors have shown that priming with either
MVA.mel3 or SFV.mel3 led to a significant expansion of NP366-374 specific
CTL upon boosting with influenza virus or MVA.mel3, respectively (FIG.
2), demonstrating that the ability to prime CTL is not unique to DNA
vectors.

[0112]In HHD A2 transgenic mice, due to the lack of CTL restricted by
endogenous mouse class I molecules, heterologous prime boost resulted in
a tremendous expansion of melan-A specific CTL up to 90% of total CD8+ T
cells (FIG. 4a), hence redirecting a large proportion of HHD mice's A2
restricted repertoire towards vaccine encoded CTL determinants. Several
factors may contribute to the immunodominance of the melan-A specific CTL
response. It is possible that a combination of an increased binding
affinity for A2 molecules and for TCR, together with a favourable
intracellular processing, may skew CTL responses towards the melan-A
epitope 26-35 in DNA.mel3 primed mice.

[0113]Previous studies have shown that "suppression" of T cell responses
specific to non dominant epitopes by T cell responses specific to
dominant epitopes is observed only when both types of determinants are
presented on the same APC32,33,38. Injection of large numbers of APC
resulted in the expansion of T cells specific to the sub-dominant
epitopes 41. The inventors have demonstrated that immunodominance of the
melan-A epitope 26-35 was overcome by boosting strategies based on the
injection of either a mixture of different recombinant viruses (FIG. 5a)
or splenocytes infected in vitro by individual vaccinia viruses encoding
full length proteins, rather than a poly-epitope construct (FIG. 5b).

Implications for Vaccination Strategies in Patients.

[0114]These results are of importance, since several clinical trials are
currently using heterologous prime boost vaccination protocols with
poly-epitope constructs. While the inventors confirm the ability of
heterologous prime-boost protocols in eliciting large numbers of vaccine
specific CTL, they demonstrate that during heterologous prime-boost
protocols, frequency of immunodominant CTL responses is significantly
expanded over frequency of CTL responses specific to less dominant
determinants. Although there are numerous mechanisms which may account
for a narrowing of the CTL repertoire which responds to a vaccine, the
inventors' results are consistent with a model of immunodominance based
on competition of T cells for antigen presenting cells (APCs) 32,33,38.
It is worth noting that the injection of vac.mel3 into naive mice induces
lymphocytocis with a; shift of CD8+ frequency from 0.5% up to 30%,
indicating a strong CTL response to the vaccinia virus. Of the CD8+ T
cells induced in the blood as many as 50% are specific for Melan-A 26-35
(data not shown), demonstrating that melan-A 26-35 is one of the most
immunodominant epitopes expressed by the virus amongst more than 200
vaccinia proteins responsible for the viruses' structure, transcription
and replication. This observation has important clinical implications for
the design of viral-based vaccines encoding the Melan-A epitope 26-35 The
use of recombinant SFV in prime-boost protocols is very attractive, as
the inventors have demonstrated that SFV can be used both as priming
vector in combination with MVA (FIG. 2) and for boosting in combination
with DNA (FIG. 4C). These results compliment a recently published report
demonstrating in macaques the enhancement of simian immunodeficiency
virus-specific immune responses induced by priming with recombinant SFV
and boosting with MVA 11.

[0115]The inventors demonstrated that pre-existing memory CTL responses
significantly reduce CTL responses specific to other epitopes contained
within the same construct (FIG. 3b). As several groups have used
immunodominant influenza peptide epitopes as positive controls during
vaccination trials, the inventors' result suggest that DNA or virus based
vaccines should not encode epitopes expressed by recurrent viruses, as
pre-existing memory CTL response may compromise the induction of CTL
responses specific to other vaccine encoded CTL determinants.

[0116]The inventors have further shown that T cell competition is likely
to play a role in modifying T cell responses in prime-boost vaccination
strategies. The inventors' work strongly suggests that simultaneous
presentation of different epitopes to a skewed repertoire of primed CTL
leads to dominant expansion of a single CTL specificity. However,
boosting the primed response with APC separately presenting the epitopes
results in comparable expansion of CTL of multiple specificities to
effective levels in vivo.

[0117]Thus, the present inventors have, amongst other things provided a
novel system for dissecting the ability of different vaccination
protocols to optimally induce polyvalent A2-restricted CTL responses. In
accordance with the present invention, methods for inducing a broad-based
CTL response should restrict the use of polyvalent constructs to the
priming phase and use separate vectors encoding individual epitopes, or
separate proteins/peptides for the boosting phase.